Outer surface proteins of Borrella burgdorferi that are essential for the bacterium to establish an infection would be excellent vaccine candidate for prevention of Lyme disease. Our laboratory has developed two methods for the identification and localization of these potential important proteins, We have developed: (1) an immune absorption technique; and (2) a membrane separation technique using equilibrium sucrose density centrifugation. With the first method, we identified ten proteins that are expressed only in low-passage, virulent B. burgdorferi strain B3I. With the second method, we have purified outer membranes of B. burgdorferL By combining these techniques, we have identified two (58- and 33-kDa) low- passage-associated outer membrane proteins. Both appear to be previously uncharacterized proteins and preliminary data suggests that one, the 58-kDa protein, may play a role in transferrin utilization by B. burgdorfen. In order to further characterize these proteins: (1) We will identify recombinant clones harboring genes encoding the 58-and 33-kDa low-passage- associated outer membrane proteins that we have previously identified. We will characterize positive clones using protein expression systems and sequence analysis. (2) We will begin the initial characterization of the 58 and 33-kDa proteins. We will construct gene fusions in order to purify recombinant 58- and 33-kDa proteins. Purified protein will be used for protection studies in mice. (3) We will isolate mutants of B. burgdorfeti that do not express low-passage-associated 58- and 33-kDa proteins and assess their role in virulence. Two recently described methods will be used: (a) the escape variant technique; and (b) gene disruption technique.